Expression Of Osteogenic Markers Research Articles

3D bio scaffold support osteogenic differentiation of mesenchymal stem cells.

The regeneration of osteochondral lesions by tissue engineering techniques is challenging due to the lack of physicochemical characteristics and dual-lineage (osteogenesis and chondrogenesis). A scaffold with better mechanical properties and dual lineage capability is required for the regeneration of osteochondral defects. In this study, a hydrogel prepared from decellularized human umbilical cord tissue was developed and evaluated for osteochondral regeneration. Mesenchymal stem cells (MSCs) isolated from the umbilical cord were seeded with hydrogel for 28 days, and cell-hydrogel composites were cultured in basal and osteogenic media. Alizarin red staining, quantitative polymerase chain reaction, and immunofluorescent staining were used to confirm that the hydrogel was biocompatible and capable of inducing osteogenic differentiation in umbilical cord-derived MSCs. The findings demonstrate that human MSCs differentiated into an osteogenic lineage following 28 days of cultivation in basal and osteoinductive media. The expression was higher in the cell-hydrogel composites cultured in osteoinductive media, as evidenced by increased levels of messenger RNA and protein expression of osteogenic markers as compared to basal media cultured cell-hydrogel composites. Additionally, calcium deposits were also observed, which provide additional evidence of osteogenic differentiation. The findings demonstrate that the hydrogel is biocompatible with MSCs and possesses osteoinductive capability in vitro. It may be potentially useful for osteochondral regeneration.

Engineered Surfaces That Promote Capture of Latent Proteins to Facilitate Integrin-Mediated Mechanical Activation of Growth Factors.

Conventional osteogenic platforms utilize active growth factors to repair bone defects that are extensive in size, but they can adversely affect patient health. Here, an unconventional osteogenic platform is reported that functions by promoting capture of inactive osteogenic growth factor molecules to the site of cell growth for subsequent integrin-mediated activation, using a recombinant fragment of latent transforming growth factor beta-binding protein-1 (rLTBP1). It is shown that rLTBP1 binds to the growth-factor- and integrin-binding domains of fibronectin on poly(ethyl acrylate) surfaces, which immobilizes rLTBP1 and promotes the binding of latency associated peptide (LAP), within which inactive transforming growth factor beta 1 (TGF-β1) is bound. rLTBP1 facilitates the interaction of LAP with integrin β1 and the subsequent mechanically driven release of TGF-β1 to stimulate canonical TGF-β1 signaling, activating osteogenic marker expression in vitro and complete regeneration of a critical-sized bone defect in vivo.

Effects of stepwise administration of osteoprotegerin and parathyroid hormone-related peptide DNA vectors on bone formation in ovariectomized rat model

Osteoporosis is a metabolic bone disease that impairs bone mineral density, microarchitecture, and strength. It requires continuous management, and further research into new treatment options is necessary. Osteoprotegerin (OPG) inhibits bone resorption and osteoclast activity. The objective of this study was to investigate the effects of stepwise administration of OPG-encoded minicircles (mcOPG) and a bone formation regulator, parathyroid hormone-related peptide (PTHrP)-encoded minicircles (mcPTHrP) in osteoporosis. The combined treatment with mcOPG and mcPTHrP significantly increased osteogenic marker expression in osteoblast differentiation compared with the single treatment groups. A model of postmenopausal osteoporosis was established in 12-week-old female rats through ovariectomy (OVX). After 8 weeks of OVX, mcOPG (80 µg/kg) was administered via intravenous injection. After 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for 3 weeks. The bone microstructure in the femur was evaluated 24 weeks after OVX using micro-CT. In a proof-of-concept study, stepwise treatment with mcOPG and mcPTHrP on an OVX rat model significantly improved bone microstructure compared to treatment with mcOPG or mcPTHrP alone. These results suggest that stepwise treatment with mcOPG and mcPTHrP may be a potential treatment for osteoporosis.

IFT80 promotes early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation as the essential functional component of primary cilia. The effects of IFT80 on early bone healing of extraction sockets have not been well studied. To investigate whether deletion of Ift80 in alveolar bone-derived mesenchymal stem cells (aBMSCs) affected socket bone healing, we generated a mouse model of specific knockout of Ift80 in Prx1 mesenchymal lineage cells (Prx1Cre ;IFT80f/f ). Our results demonstrated that deletion of IFT80 in Prx1 lineage cells decreased the trabecular bone volume, ALP-positive osteoblastic activity, TRAP-positive osteoclastic activity, and OSX-/COL I-/OCN-positive areas in tooth extraction sockets of Prx1Cre ; IFT80f/f mice compared with IFT80f/f littermates. Furthermore, aBMSCs from Prx1Cre ; IFT80f/f mice showed significantly decreased osteogenic markers and downregulated migration and proliferation capacity. Importantly, the overexpression of TAZ recovered significantly the expressions of osteogenic markers and migration capacity of aBMSCs. Lastly, the local administration of lentivirus for TAZ enhanced the expression of RUNX2 and OSX and promoted early bone healing of extraction sockets from Prx1Cre ; IFT80f/f mice. Thus, IFT80 promotes osteogenesis and early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Enhancing osteogenic differentiation of dental pulp stem cells through rosuvastatin loaded niosomes optimized by Box-Behnken design and modified by hyaluronan: a novel strategy for improved efficiency

Bone tissue engineering necessitates a stem cell source capable of osteoblast differentiation and mineralized matrix production. Dental pulp stem cells (DPSCs), a subtype of mesenchymal stem cells from human teeth, present such potential but face challenges in osteogenic differentiation. This research introduces an innovative approach to bolster DPSCs’ osteogenic potential using niosomal and hyaluronan modified niosomal systems enriched with rosuvastatin. While rosuvastatin fosters bone formation by regulating bone morphogenetic proteins and osteoblasts, its solubility, permeability, and bioavailability constraints hinder its bone regeneration application. Using a Box-Behnken design, optimal formulation parameters were ascertained. Both niosomes were analyzed for size, polydispersity, zeta potential, and other parameters. They displayed average sizes under 275 nm and entrapment efficiencies exceeding 62%. Notably, niosomes boosted DPSCs’ cell viability and osteogenic marker expression, suggesting enhanced differentiation and bone formation. Conclusively, the study underscores the potential of both niosomal systems in ameliorating DPSCs’ osteogenic differentiation, offering a promising avenue for bone tissue engineering and regeneration.Graphical

Dopamine-mediated copper-loaded ZnTiO3 antimicrobial coating with immunomodulatory properties effectively enhances vascularised osteogenesis on titanium implants

In this study, zinc (Zn) with osteogenic activity and copper (Cu) with angiogenic activity were simultaneously introduced into titanium dioxide nanotubes (TN) by means of anodic oxidation technique, hydrothermal reaction and dopamine (PDA) polymerisation effect (ZnTN-PDA-Cu). The ZnTN-PDA-Cu coating had favourable mechanical, hydrophilic, corrosion-resistant, and bioactive characteristics, according to the results. Zn2+ and Cu2+ could be continuously released from the coating and was pH-responsive. In addition, ZnTN-PDA-Cu showed good antibacterial properties against E. coli and S. aureus. Cellular assays revealed that RAW264.7 (immune), HUVEC (angiogenic) and MC3T3-E1 (osteogenic) exhibited good adhesion and proliferation activities on the ZnTN-PDA-Cu coated Ti. The expression of anti-inflammatory markers, angiogenic markers and osteogenic markers was significantly up-regulated in ZnTN-PDA-Cu coatings at both the gene level and protein level. ZnTN-PDA-Cu also demonstrated the capacity to stimulate RAW264.7 towards M2 polarisation and HUVEC tube formation. In addition, it demonstrated the capacity to stimulate MC3T3-E1 towards osteogenic differentiation. In summary, the ZnTN-PDA-Cu coating had better antibacterial, anti-inflammatory, angiogenic and osteoinductive properties. The multifunctional coatings prepared in this study provide a promising new strategy and experimental basis for the design of antibacterial titanium surfaces with immunomodulatory functions.

Piezo1 contributes to alveolar bone remodeling by activating β-catenin under compressive stress

The mechanosensitive ion channel, Piezo1, is responsible for transducing mechanical stimuli into intracellular biochemical signals and has been identified within periodontal ligament cells (PDLCs). Nonetheless, the precise biologic function of Piezo1 in the regulation of alveolar bone remodeling by PDLCs during compressive forces remains unclear. Therefore, this study focused on elucidating the role of the Piezo1 channel in alveolar bone remodeling and uncovering its underlying mechanisms. PDLCs were subjected to compressive force and Piezo1 inhibitors. Piezo1 and β-catenin expressions were quantified by quantitative reverse transcription polymerase chain reaction and Western blot. The intracellular calcium concentration was measured using Fluo-8 AM staining. The osteogenic and osteoclastic activities were assessed using alkaline phosphatase staining, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and Western blot. Invivo, orthodontic tooth movement was used to determine the effects of Piezo1 on alveolar bone remodeling. Piezo1 and activated β-catenin expressions were upregulated under compressive force. Piezo1 inhibition reduced β-catenin activation, osteogenic differentiation, and osteoclastic activities. β-catenin knockdown reversed the increased osteogenic differentiation but had little impact on osteoclastic activities. Invivo, Piezo1 inhibition led to decreased tooth movement distance, accompanied by reduced β-catenin activation and expression of osteogenic and osteoclastic markers on the compression side. The Piezo1 channel is a key mechanotransduction component of PDLCs that senses compressive force and activates β-catenin to regulate alveolar bone remodeling.

Pten knockout in mouse preosteoblasts leads to changes in bone turnover and strength.

Bone development and remodeling are controlled by the phosphoinositide-3-kinase (Pi3k) signaling pathway. We investigated the effects of downregulation of phosphatase and tensin homolog (Pten), a negative regulator of Pi3k signaling, in a mouse model of Pten deficiency in preosteoblasts. We aimed to identify mechanisms that are involved in the regulation of bone turnover and are linked to bone disorders. Femora, tibiae, and bone marrow stromal cells (BMSCs) isolated from mice with a conditional deletion of Pten (Pten cKO) in Osterix/Sp7-expressing osteoprogenitor cells were compared to Cre-negative controls. Bone phenotyping was performed by μCT measurements, bone histomorphometry, quantification of bone turnover markers CTX and procollagen type 1N propeptide (P1NP), and three-point bending test. Proliferation of BMSCs was measured by counting nuclei and Ki-67-stained cells. In vitro, osteogenic differentiation capacity was determined by ALP staining, as well as by detecting gene expression of osteogenic markers. BMSCs from Pten cKO mice were functionally different from control BMSCs. Osteogenic markers were increased in BMSCs derived from Pten cKO mice, while Pten protein expression was lower and Akt phosphorylation was increased. We detected a higher trabecular bone volume and an altered cortical bone morphology in Pten cKO bones with a progressive decrease in bone and tissue mineral density. Pten cKO bones displayed fewer osteoclasts and more osteoblasts (P = .00095) per trabecular bone surface and a higher trabecular bone formation rate. Biomechanical analysis revealed a significantly higher bone strength (P = .00012 for males) and elasticity of Pten cKO femora. On the cellular level, both proliferation and osteogenic differentiation capacity of Pten cKO BMSCs were significantly increased compared to controls. Our findings suggest that Pten knockout in osteoprogenitor cells increases bone stability and elasticity by increasing trabecular bone mass and leads to increased proliferation and osteogenic differentiation of BMSCs.

Effect of exogenous genistein on osteogenic differentiation of adipose-derived mesenchymal stem cells in laying hens

Previous literature revealed that genistein might play a preventive role in osteoporosis. Therefore, we aimed to evaluate the effect of genistein on the osteogenic potency of laying hens’ adipose-derived stem cells (LHASCs). The viability of LHASCs after isolation was investigated on tissue culture plastic (TCP) under exposure to genistein up to 50 μg/mL by MTT assay. Our preliminary result revealed that LHASCs cultured under genistein exposure up to 20 μg/mL are feasible. Then, we evaluated the osteogenic induction of LHASCs under exposure to 0, 10, and 20 μg/mL genistein. The Alizarin Red staining confirmed the calcium deposition. Our findings showed that osteogenic differentiation under exposure to 20 μg/mL genistein led to higher ALP activity and more calcium content. We then tried to see the probable additive effect of the genistein-plus Poly-L-lactic acid (PLLA) scaffold on the cell viability and osteogenic capacity of LHASCs. For this, cells were cultured on a PLLA scaffold and exposed to 20 μg/mL genistein. Cell growth rate, as indicated by the MTT assay, revealed no differences between the groups. LHASCs cultured on a genistein-plus PLLA scaffold showed higher ALP activity and more calcium content. The expressions of Osteocalcin, COL1A2, ALP, and Runx2 genes were increased in the genistein-plus PLLA group as compared with PLLA and TCP groups. Adequate proliferation rates and higher expression of osteogenic markers provide genistein as a suitable substrate to support the proliferation and differentiation of LHASCs. Genistein supports osteogenic induction as a further positive effect if such a PLLA scaffold is available.

Effect of a synthetic hydroxyapatite-based bone grafting material compared to established bone substitute materials on regeneration of critical-size bone defects in the ovine scapula.

Lately, the potential risk of disease transmission due to the use of bovine-derived bone substitutes has become obvious, demonstrating the urgent need for a synthetic grafting material with comparable bioactive behaviour and properties. Therefore, the effect of a synthetic hydroxyapatite (HA) (Osbone®) bone grafting material on bone regeneration was evaluated 2 weeks, 1 month, and 3, 6, 12 and 18 months after implantation in critical-size bone defects in the ovine scapula and compared to that of a bovine-derived HA (Bio-Oss®) and β-tricalcium phosphate (TCP) (Cerasorb® M). New bone formation and the biodegradability of the bone substitutes were assessed histomorphometrically. Hard tissue histology and immunohistochemical analysis were employed to characterize collagen type I, alkaline phosphatase, osteocalcin, as well as bone sialoprotein expression in the various cell and matrix components of the bone tissue to evaluate the bioactive properties of the bone grafting materials. No inflammatory tissue response was detected with any of the bone substitute materials studied. After 3 and 6 months, β-TCP (Cerasorb® M) showed superior bone formation when compared to both HA-based materials (3 months: β-TCP 55.65 ± 2.03% vs. SHA 49.05 ± 3.84% and BHA 47.59 ± 1.97%; p ≤ 0.03; 6 months: β-TCP 62.03 ± 1.58%; SHA: 55.83 ± 2.59%; BHA: 53.44 ± 0.78%; p ≤ 0.04). Further, after 12 and 18 months, a similar degree of bone formation and bone-particle contact was noted for all three bone substitute materials without any significant differences. The synthetic HA supported new bone formation, osteogenic marker expression, matrix mineralization and good bone-bonding behaviour to an equal and even slightly superior degree compared to the bovine-derived HA. As a result, synthetic HA can be regarded as a valuable alternative to the bovine-derived HA without the potential risk of disease transmission.

NOVEL PIEZOELECTRIC AND OSTEOCONDUCTIVE NANOFIBRES FOR BONE TISSUE ENGINEERING

The current procedures being applied in the clinical setting to address osteoporosis-related delayed union and nonunion bone fractures have been found to present mostly suboptimal outcomes. As a result, bone tissue engineering (BTE) solutions involving the development of implantable biomimetic scaffolds to replace damaged bone and support its regeneration are gaining interest. The piezoelectric properties of the bone tissue, which stem primarily from the significant presence of piezoelectric type I collagen fibrils in the tissue's extracellular matrix (ECM), play a key role in preserving the bone's homeostasis and provide integral assistance to the regeneration process. However, despite their significant potential, these properties of bone tend to be overlooked in most BTE-related studies. In order to bridge this gap in the literature, novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) electrospun nanofibers were developed to replicate the bone's fibrous ECM composition and electrical features. Different HAp nanoparticle concentrations (1–10%, wt%) were tested to assess their effect on the physicochemical and biological properties of the resulting fibers. The fabricated scaffolds displayed biomimetic collagen fibril-like diameters, while also presenting mechanical features akin to type I collagen. The increase in HAp presence was found to enhance both surface and piezoelectric properties of the fibers, with an improvement in scaffold wettability and increase in β-phase nucleation (translating to increased piezoelectricity) being observed. The HAp-containing scaffolds also exhibited an augmented bioactivity, with a more comprehensive surface mineralization of the fibers being obtained for the scaffolds with the highest HAp concentrations. Improved osteogenic differentiation of seeded human mesenchymal stem/stromal cells was achieved with the addition of HAp, as confirmed by an increased ALP activity, calcium deposition and upregulated expression of key osteogenic markers. Overall, our findings highlight, for the first time, the potential of combining PVDF-TrFE and HAp to develop electroactive and osteoinductive nanofibers for BTE.Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), the PhD scholarship (2022.10572.BD) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020).

NOVEL MINERALIZED ELECTROACTIVE PAN/PEDOT:PSS NANOFIBERS FOR BONE TISSUE ENGINEERING

Bone defects can result from different incidents such as acute trauma, infection or tumor resection. While in most instances bone healing can be achieved given the tissue's innate ability of self-repair, for critical-sized defects spontaneous regeneration is less likely to occur, therefore requiring surgical intervention. Current clinical procedures have failed to adequately address this issue. For this reason, bone tissue engineering (BTE) strategies involving the use of synthetic grafts for replacing damaged bone and promoting the tissue's regeneration are being investigated. The electrical stimulation (ES) of bone defects using direct current has yielded very promising results, with neo tissue formation being achieved in the target sites in vivo. Electroactive implantable scaffolds comprised by conductive biomaterials could be used to assist this kind of therapy by either directing the ES specifically to the damaged site or promoting the integration of electrodes within the bone tissue as a coating. In this study, we developed novel conductive heat-treated polyacrylonitrile/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PAN/PEDOT:PSS) nanofibers via electrospinning capable of mimicking key native features of the bone tissue's extracellular matrix (ECM) and providing a platform for the delivery of exogenous ES. The developed scaffolds were doped with sulfuric acid and mineralized in Simulated Body Fluid to mimic the inorganic phase of bone ECM. As expected, the doped PAN/PEDOT:PSS nanofibers exhibited electroconductive properties and were able to preserve their fibrous structure. The addition of PEDOT:PSS was found to improve the bioactivity of the scaffolds, with a more significant in vitro mineralization being obtained. By seeding the scaffolds with MG-63 osteoblasts and human mesenchymal stem/stromal cells, an increased cell proliferation was observed for the mineralized PAN/PEDOT:PSS nanofibers, which also registered an increased expression of key osteogenic markers (e.g Osteopontin). Our findings appear to corroborate the promising potential of the generated nanofibers for future ES-based BTE applications.Acknowledgements: The authors thank FCT for funding through the projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), BioMaterARISES (EXPL/CTM-CTM/0995/2021) and OptiBioScaffold (PTDC/EME-SIS/32554/2017, POCI-01- 0145-FEDER- 32554), the PhD scholarship (2022.10572.BD) and through institutional funding to iBB (UIDB/04565/2020 and UIDP/04565/2020), Associate Laboratory i4HB (LA/P/0140/2020) and IT (UIDB/50008/2020).

3D POROUS COMPOSITE SCAFFOLDS WITH HIGH CALCIUM PHOSPHATE CONTENT AND ADDITION OF INORGANIC IONS FOR BONE REGENERATION APPLICATIONS

Critical-sized bone defects remain challenging in the clinical setting. Autologous bone grafting remains preferred by clinicians. However, the use of autologous tissue is associated with donor-site morbidity and limited accessibility to the graft tissue. Advances in the development of synthetic bone substitutes focus on improving their osteoinductive properties. Whereas osteoinductivity has been demonstrated with ceramics, it is still a challenge in case of polymeric composites. One of the approaches to improve the regenerative properties of biomaterials, without changing their synthetic character, is the addition of inorganic ions with known osteogenic and angiogenic properties. We have previously reported that the use of a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the TCP particles can enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) (3). To further support the regenerative properties of these scaffolds, inorganic ions with known angiogenic properties, copper or cobalt, were added to the coating solution.β-TCP particles were immersed in a zinc and copper or zinc and cobalt solution with a concentration of 15 or 45 mM. 3D porous scaffolds composed of 1000PEOT70PBT30 and pure or coated β-TCP were additively manufactured by 3D fibre deposition. The osteogenic and angiogenic properties of the fabricated scaffolds were tested in vitro through culture with hMSCs and human umbilical vein endothelial cells, respectively. The materials were further evaluated through ectopic implantation in an in vivo mini-pig model. The early expression of relevant osteogenic gene markers (collagen-1, osteocalcin) of hMSCs was upregulated in the presence of lower concentration of inorganic ions. Further analysis will focus on the evaluation of ectopic bone formation and vascularisation of these scaffolds after implantation in a mini-pig ectopic intramuscular model.

CXXC5 mitigates P. gingivalis-inhibited cementogenesis by influencing mitochondrial biogenesis

BackgroundCementoblasts on the tooth-root surface are responsible for cementum formation (cementogenesis) and sensitive to Porphyromonas gingivalis stimulation. We have previously proved transcription factor CXXC-type zinc finger protein 5 (CXXC5) participates in cementogenesis. Here, we aimed to elucidate the mechanism in which CXXC5 regulates P. gingivalis-inhibited cementogenesis from the perspective of mitochondrial biogenesis.MethodsIn vivo, periapical lesions were induced in mouse mandibular first molars by pulp exposure, and P. gingivalis was applied into the root canals. In vitro, a cementoblast cell line (OCCM-30) was induced cementogenesis and submitted for RNA sequencing. These cells were co-cultured with P. gingivalis and examined for osteogenic ability and mitochondrial biogenesis. Cells with stable CXXC5 overexpression were constructed by lentivirus transduction, and PGC-1α (central inducer of mitochondrial biogenesis) was down-regulated by siRNA transfection.ResultsPeriapical lesions were enlarged, and PGC-1α expression was reduced by P. gingivalis treatment. Upon apical inflammation, Cxxc5 expression decreased with Il-6 upregulation. RNA sequencing showed enhanced expression of osteogenic markers, Cxxc5, and mitochondrial biogenesis markers during cementogenesis. P. gingivalis suppressed osteogenic capacities, mitochondrial biogenesis markers, mitochondrial (mt)DNA copy number, and cellular ATP content of cementoblasts, whereas CXXC5 overexpression rescued these effects. PGC-1α knockdown dramatically impaired cementoblast differentiation, confirming the role of mitochondrial biogenesis on cementogenesis.ConclusionsCXXC5 is a P. gingivalis-sensitive transcription factor that positively regulates cementogenesis by influencing PGC-1α-dependent mitochondrial biogenesis.A5vAuiTS6Tkq1RfziX_nr_Video

Iron oxide nanoparticle-stabilized Pickering emulsion-templated porous scaffolds loaded with polyunsaturated fatty acids (PUFAs) for bone tissue engineering.

Dietary intake of ω-3-polyunsaturated fatty acids (PUFAs) can significantly improve the expression levels of alkaline phosphatase (ALP) and osteocalcin. However, PUFAs are hydrophobic and highly sensitive to temperature, oxygen concentration, pH, and ionic strength. Hence, it is challenging to use PUFAs as bioactive compounds for bone tissue engineering. Here, we encapsulated PUFAs in liposomes to improve their stability. The hydrodynamic size of the PUFA-loaded liposomes was found to be 121.3 ± 35 nm. GC-MS analysis showed that the encapsulation efficiency of the PUFAs was 19.9 ± 3.4%. These PUFA-loaded liposomes were loaded into porous scaffolds that were prepared by polymerizing glycidyl methacrylate and trimethylolpropane triacrylate monomers using the Pickering emulsion polymerization technique. Oleic acid-coated iron oxide nanoparticles were used as the stabilizing agent to prepare these acrylate-based scaffolds containing PUFA-loaded liposomes (P-Lipo-IO(GMA-TMPTA)). SEM micrographs confirmed the porous nature of the scaffolds and the presence of well-adhered liposomes. An in vitro cytotoxicity study conducted using MG63 cells confirmed that these scaffolds showed desirable cytocompatibility. Cell adhesion study showed a well-spread morphology, indicating firm adhesion of the cells. The alizarin red staining of P-Lipo-IO(GMA-TMPTA) scaffolds showed 3- and 2-fold higher calcium deposition compared to the control on days 7 and 14, respectively. ALP activity was also 2-fold higher than that of the control on day 14. RT-PCR analysis of cells exposed to P-Lipo-IO(GMA-TMPTA) scaffolds showed significantly higher expression of osteogenic markers compared to the control. An antibacterial study conducted on Staphylococcus aureus showed a higher percentage inhibition and reactive oxygen species generation in samples treated with P-Lipo-IO(GMA-TMPTA) scaffolds. These desirable biological properties indicate that the developed scaffolds are suitable for bone tissue engineering.

PCAT-1' s role in wound healing impairment: Mitochondrial dysfunction and bone marrow stem cell differentiation inhibition via PKM2/β-catenin pathway and its impact on implant osseo-integration.

This study focused on unravelling the role of PCAT-1 in wound-healing process, particularly its impact on regenerative and osteogenic abilities of mesenchymal stem cells (MSCs). We delved into how PCAT-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) and interacts with pivotal molecular pathways, especially β-catenin and PKM2, using human bone marrow-derived MSCs. MSCs were cultured under specific conditions and PCAT-1 expression was modified through transfection. We thoroughly assessed several critical parameters: MSC proliferation, mitochondrial functionality, ATP production and expression of wound healing and osteogenic differentiation markers. Further, we evaluated alkaline phosphatase (ALP) activity and mineral deposition, essential for bone healing. Our findings revealed that overexpressing PCAT-1 significantly reduced MSC proliferation, hampered mitochondrial performance and lowered ATP levels, suggesting the clear inhibitory effect of PCAT-1 on these vital wound-healing processes. Additionally, PCAT-1 overexpression notably decreased ALP activity and calcium accumulation in MSCs, crucial for effective bone regeneration. This overexpression also led to the reduction in osteogenic marker expression, indicating suppression of osteogenic differentiation, essential in wound-healing scenarios. Moreover, our study uncovered a direct interaction between PCAT-1 and the PKM2/β-catenin pathway, where PCAT-1 overexpression intensified PKM2 activity while inhibiting β-catenin, thereby adversely affecting osteogenesis. This research thus highlights PCAT-1's significant role in impairing wound healing, offering insights into the molecular mechanisms that may guide future therapeutic strategies for enhancing wound repair and bone regeneration.

Ginsenoside Rg1 modulates PI3K/AKT pathway for enhanced osteogenesis via GPER

BackgroundOsteoporosis is a systemic skeletal disorder characterized by decreased bone density and the degradation of bone tissue microarchitecture. Ginsenoside Rg1, derived from Panax ginseng, has been a part of traditional Chinese medicine in China for centuries, particularly for treating osteoporosis. However, there remains limited research on the osteogenic potential of Rg1 within the glucocorticoid-induced osteoporosis (GIOP) model and its specific mechanisms. PurposeThe primary objective of this study is to investigate the osteogenic potential of Rg1 within the GIOP model and explore the signaling pathways associated with its in vivo and in vitro effects. MethodsCell proliferation, differentiation and mineralization were evaluated by the Cell counting kit 8(CCK8) assay, alkaline phosphatase (ALP) test and Alizarin Red S staining, respectively. The qPCR technique was used to determine the relative expression of mRNA and the western blot was used to determine the relative expression of protein. In vivo experiments, spinal vertebrae staining in zebrafish larvae was accomplished by alizarin red S staining. ResultsZebrafish larvae's hatching, survival, malformation, and heart rate were unaffected by 50 μM of Rg1 in vivo, while the MEC3T3-E1 cell line's proliferation was unaffected by 50 μM of Rg1 in vitro. Meanwhile, Rg1 was shown to improve osteogenic differentiation or bone formation as well as the level of mRNA expression of osteogenic markers in vivo and in vitro. Treatment with Rg1 significantly increased the expression of G protein-coupled estrogen receptor (GPER) and pAKT. In addition, the GPER inhibitor G15 could significantly reduce the mRNA and protein expression levels of GPER and phosphorylated AKT, LY294002, a PI3K/AKT pathway inhibitor, markedly suppresses the expression of phosphorylated AKT, yet shows no significant impact on GPER expression. Both G15 and LY294002 can significantly blocked the Rg1-mediated enhancement of osteogenesis capacity in the GIOP model. In contrast, when both the agonists G1 of GPER and LY294002 were added, G1 increased the relative expression of mRNA and protein of GPER, but not the expression of osteogenic capacity and osteogenic markers. ConclusionsThis study investigates the mineralization effects and mechanisms of Ginsenoside Rg1 both in vitro and in vivo. For the first time, we propose that Rg1 might regulate osteogenesis by modulating AKT phosphorylation through mediating GPER expression within the PI3K/AKT pathway in the GIOP model. This discovery introduces novel targets and avenues for osteoporosis treatment.

Osteogenic Effect of a Bioactive Calcium Alkali Phosphate Bone Substitute in Humans.

(1) Background: The desire to avoid autograft harvesting in implant dentistry has prompted an ever-increasing quest for bioceramic bone substitutes, which stimulate osteogenesis while resorbing in a timely fashion. Consequently, a highly bioactive silicon containing calcium alkali orthophosphate (Si-CAP) material was created, which previously was shown to induce greater bone cell maturation and bone neo-formation than β-tricalcium phosphate (β-TCP) in vivo as well as in vitro. Our study tested the hypothesis that the enhanced effect on bone cell function in vitro and in sheep in vivo would lead to more copious bone neoformation in patients following sinus floor augmentation (SFA) employing Si-CAP when compared to β-TCP. (2) Methods: The effects of Si-CAP on osteogenesis and Si-CAP resorbability were evaluated in biopsies harvested from 38 patients six months after SFA in comparison to β-TCP employing undecalcified histology, histomorphometry, and immunohistochemical analysis of osteogenic marker expression. (3) Results: Si-CAP as well as β-TCP supported matrix mineralization and bone formation. Apically furthest away from the original bone tissue, Si-CAP induced significantly higher bone formation, bone-bonding (bone-bioceramic contact), and granule resorption than β-TCP. This was in conjunction with a higher expression of osteogenic markers. (4) Conclusions: Si-CAP induced higher and more advanced bone formation and resorbability than β-TCP, while β-TCP's remarkable osteoconductivity has been widely demonstrated. Hence, Si-CAP constitutes a well-suited bioactive graft choice for SFA in the clinical arena.

Niacin treatment prevents bone loss in iron overload osteoporotic rats via activation of SIRT1 signaling pathway

Recently, more and more studies have revealed that iron overload can lead to osteoporosis by inducing oxidative stress. Niacin (NAN), also known as nicotinate or vitamin B3, has been confirmed to possess potent antioxidative effects. In addition, very little is currently known about the protective effects of NAN on iron overload in osteoporotic bone tissue. Therefore, we aimed to evaluate the protective effect of niacin on iron overload-induced bone injury and to investigate the effect and underlying mechanisms of the niacin and iron overload on intracellular antioxidant properties. When MC3T3-E1 and RAW264.7 cells were cultured in the presence of ammonium ferric citrate(FAC), NAN therapy could increase the matrix mineralization and promote expression of osteogenic markers in MC3T3-E1, inhibit osteoclastic differentiation of RAW264.7 cells, while increasing intracellular reactive oxygen species (ROS) levels and strengthening mitochondrial membrane potential (MMP). In the ovariectomized (OVX) rat model, NAN had an obvious protective effect against iron-overloaded injury. Meanwhile, superoxide dismutase 2 (SOD2), intracellular antioxidant enzymes and silent information regulator type 1 (SIRT1), were up-regulated in response to NAN exposures in MC3T3-E1. Furthermore, SIRT1 inhibitor EX527 attenuated the protective effects of NAN. Results revealed that NAN could stimulate osteogenic differentiation, inhibit osteoclastic differentiation and markedly increased antioxidant properties in cells through the induction of SIRT1. Studies suggest that niacin is a promising agent for preventing bone loss in iron overload conditions.

Effect of pulp capping materials on odontogenic differentiation of human dental pulp stem cells: An in vitro study.

Migration and differentiation of human dental pulp stem cells (hDPSCs) is a vital and key factor in the success of reparative dentin formation for maintenance of pulp vitality. Pulp capping materials are used to stimulate DPSCs to induce new dentin formation. Thus, the aim of the present study was to compare the response of DPSCs to four commercially available pulp capping materials: a bioactive bioceramic (Material 1), a nonresinous ready-to-use bioceramic cement (Material 2), a bioactive composite (Material 3),and a biocompatible, dual-cured, resin-modified calcium silicate (Material 4). hDPSCs were isolated and cultured from freshly extracted teeth and were then characterized by flow cytometry and multilineage differentiation. Discs prepared from pulp capping materials were tested with hDPSCs and MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, cell migration assay and odontogenic differentiation assay was performed. Expression of osteogenic markers (osteopontin, RUNX family transcription factor 2, osteocalcin) and the odontogenic marker (dentin sialophosphoprotein) was detected using reverse transcription-polymerase chain reaction. Materials 1, 2, and 3 generated more cell viability than Material 4. Furthermore, Material 4 showed the least wound exposure percentage, while Material 3 showed the highest percentage. Enhanced mineralization was found in hDSCPs cultured with Material 3,followed by Material 1, and then Material 2, while Material 4 revealed the least calcified mineralization. The results of this study were inconclusive regards contemporary bioceramic materials designed for vital pulp therapy as they have different effects on hDPSC. Further testing for cytotoxicity using live-dead staining, animal experiments, clinical trials, and independent analyses of these biomaterials is necessary for clinicians to make an informed decision for their use.